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Folia Morphol. Vol. 73, No. 4, pp. 439–448 DOI: 10.5603/FM.2014.0066 O R I G I N A L A R T I C L E Copyright © 2014 Via Medica ISSN 0015–5659 www.fm.viamedica.pl The stratigraphical organisation of the micro vascular systems of the porcine vocal folds H. Reinhard, A. Lang, H. Gasse Institute of Anatomy, University of Veterinary Medicine Hannover, Germany [Received 27 February 2014; Accepted 14 April 2014] The cranial and caudal vocal folds (CraF, CauF) of the glottis of adult minipigs (11–27 months; n = 12) were examined after immunohistochemical application of polyclonal anti-von-Willebrand-Factor and anti-Smooth-Muscle-Actin in serial paraffin sections. This examination aimed at a stratigraphical analysis of microvessels; data were compared with findings in humans which had been reported in the literature. (1) The distribution of the microvessels was very heterogeneous in the CraF and in the CauF, but a common pattern existed in both. (2) Characteristic vascular zones and rows were detected; each of them displayed a specific distribution and density of blood capillaries, arterioles, venules, lymphatic capillaries, and lymphatic precollectors. (3) A striking feature was the presence of a subepithelial Avascu- lar Band and of a focal Avascular Area within the lamina propria of the fold’s crests. (4) The vascular zones, the rows, the Avascular Band, and the Avascular Area could be allocated to specific layers of the lamina propria: subepithelial, superficial, intermediate, deep layer. (5) The loose Avascular Area at the level of the superficial layer of the lamina propria (in both CraF and CauF) corresponded to Reinke’s space in humans in terms of structure and location. (6) The direction/ /course of blood and lymphatic microvessels shared common features with that of the human vocal fold. (Folia Morphol 2014; 73, 4: 439–448) Key words: anti-von-Willebrand-Factor, anti-Smooth-Muscle-Actin, lymphatics, paraffin sections INTRODUCTION trol the viscosity of the vocal fold’s tissue [6]. In terms The viscoelastic properties of the vocal folds have of this, the mechanisms of inflow, binding (stasis), a major impact on the vibratory movement during and outflow of fluids deserve major attention. In this phonation [9]. These properties are related to se- paper, stress shall be put on the structural component veral features: Firstly, they depend on the amount of these systems, i.e. blood capillaries and lymphatic and distribution of collagenous and elastic fibres, capillaries/lymphatic precollectors. The description of which characterise the stratigraphical organisation their local distribution shall complement the detailed of the lamina propria [3, 11, 49]; secondly, they are stratigraphical examinations of the minipig’s vocal influenced by the local distribution of the amorphous folds [17], whose lamina propria is subdivided into Ground Substance [26]. 4 layers: subepithelial, superficial, intermediate, and The interstitial fluid in the Ground Substance and, deep layer. These layers are characterised by different in particular, the molecules in it affect and often con- widths, fibre qualities, and fibre densities. Address for correspondence: Dr H. Gasse, Institute of Anatomy, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany, tel: 0511 8567573, fax: 0511 856827214, e-mail: [email protected] 439 Folia Morphol., 2014, Vol. 73, No. 4 In particular, band-like or focal regions, which were conspicuous because of their sparse number of fibres [17] and blood vessels, attracted special attention, as they may be related to Reinke’s space in the human glottis. In humans, Reinke’s space may become frequently affected by pathological condi- tions, especially by Reinke’s oedema [15, 32, 33, 35], which influences the voice [8]. In the human vocal fold, the lymph vascular system [16, 34, 18–20, 44–47] and the blood vascular system [23, 25, 34] have been studied intensively. However, Figure 1. Bisected porcine larynx; a — cranial fold; b — caudal topographical and stratigraphical relations have not fold; the opening of the laryngeal ventricle lying in-between; been fully considered. Consequently, the location of c — position of the vestibular ligament. the different microvascular networks in situ and their attribution to certain histological layers of the lamina propria still have to be evaluated. This is even more (saturated aqueous picric acid, filtered acid-free form- relevant because of the species-specific anatomical aldehyde 37%, acetic acid 98%; 25:10:1) at room characteristic of the pig’s glottis. A distinction between temperature for 48 h. For histological processing, the its phoniatrically relevant cranial and caudal fold (for larynx was bisected in the median plane. Then, the details see [17]) is mandatory, especially if the minipig glottis was excised from each half and cut transver- is intended to be used as a model for humans. sely at its midportion (Fig. 1). These specimens were A lightmicroscopical approach on tissue sections then treated with the standard procedure of alcoholic from the minipig was used in order to evaluate the dehydration and paraffin embedding. Serial cross topographical distribution simultaneously of both, sections (5–8 µm) were alternately stained either with blood and lymph vascular structures in situ. This was performed by the immunohistochemical application Masson’s Trichrome or haematoxylin and eosin. Four of polyclonal anti-von-Willebrand-Factor (anti-SMA) other sections were immunostained with polyclonal and anti-Smooth-Muscle-Actin (anti-vWF). anti-vWF. Another 4 sections were immunostained with monoclonal anti-SMA Clone 1A4. One section MATERIALS AND METHODS of each immunostain was selected for the histomor- The specimens were taken from the larynges of fe- phometrical examination. All sections represented male minipigs (aged 11–27 months, n = 12) of the the midportion of the folds. Göttinger Minipig and Mini Lewe Minipig breeds. All Immunohistochemical staining: anti-vWF, anti-SMA animals were obtained and euthanased primarily for the anatomical dissection classes at the University of The paraffin cross sections were deparaffinised Veterinary Medicine Hannover. All related procedures in xylol and rehydrated in a graded alcohol series. were performed in accordance with the Guidelines of Endogenous peroxidase was inhibited using 0.6% the European Convention for the Protection of Verte- hydrogen peroxide in alcohol 80% for 30 min. Three brate Animals used for Experimental and other Scientific washes (5 min each) were performed with 0.05 M Purposes (86/609/EEC). Respecting this, an approval by trisphosphate-buffered saline with Tween TBS-T pH the Lower Saxony State Office for Consumer Protec- 7.4 between each step of the procedure, if not other- tion and Food Safety was not necessary. The animals wise stated. Antigen retrieval was performed diffe- were euthanased either by injection (by a veterinarian) of rently: the sections designed to be immunostained 1.5 mL/10 kg Euthadorm® (pentobarbital sodium) or with anti-vWF were pretreated with proteinase K by captive bolt stunning and consecutive bleeding; they (Sigma-Aldrich) in phosphate-buffered saline 1:200 showed no signs of larynx-associated diseases upon at 37°C for 25 min; the sections designed to be im- examination. munostained with anti-SMA were placed in a water bath containing citric buffer (pH 6.0) and heated at Histological procedures 95–99°C for 20 min. In order to prevent non-specific The larynges were excised immediately after binding of antibodies, all sections were treated with euthanasia and immersion-fixed in Bouin’s solution normal goat serum at room temperature for 20 min. 440 H. Reinhard et al., Microvascular systems of the porcine vocal folds A B Figure 2. Immunohistochemical staining of two adjacent serial paraffin cross sections of the porcine glottis; A. Anti-von-Willebrand-Factor detected endothelium of blood vessels (arrowheads) and endothelium of initial lymphatics (arrows); B. Anti-Smooth-Muscle-Actin (anti-SMA) detected blood vessels (arrowheads), but no initial lymphatics (arrows: anti-SMA negative lymph capillary). Table 1. Immunoreactivity of anti-von-Willebrand-Factor (anti-vWF) and anti-Smooth-Muscle-Actin (anti-SMA) in vascular cells, as recorded in the literature Blood vascular endothelium Lymph vascular endothelium Smooth muscle cells Pericytes Anti-vWF [13, 28, 30] + + – – Anti-SMA [27, 28, 38, 39] – – + + Primary antibodies were applied for 16–18 h at 4–8°C Histomorphometric examination of the microvessels at a dilution of 1:400 (anti-vWF; Dako, Hamburg, The stained cross sections were digitised (‘Scan Germany) or at a dilution of 1:100 (anti-SMA Clone Scope’ Aperio) and subsequently histomorphometri- 1A4; Dako, Hamburg, Germany). The sections were cally examined with the aid of the graphics editing then incubated with Dako EnVision + System/HRP program Adobe Photoshop CS 3 Extended 10.0.1 (a dextran polymer with secondary antibody and (Adobe Systems). In a preliminary study [31], a com- horseradish peroxidase; Dako, Hamburg, Germany) mon pattern of vascular distribution had been found for 30 min. The reaction was visualised by exposure for both the cranial and caudal vocal folds (CraF, to diaminobenzidine in a substrate buffer (Dako, CauF). This pattern was used to make a stratigraphical Hamburg, Germany). Afterwards, the sections were subdivision of the examined tissue areas. The key fea- washed 3 times (5 min) with phosphate buffered tures of this common pattern,

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